In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol
Abstract
:1. Introduction
2. Results
2.1. Optical Absorption Spectroscopy
2.2. Influence of Buffer and Glycerol on CW EPR Spectra
2.3. Subtle Changes in the Heme Pocket Revealed by Pulsed EPR
3. Discussion
3.1. Heme Cavity Heterogeneity and the Influence of Glycerol
3.2. The Non-Innocent Effect of Buffer Molecules
3.3. Alkaline Transition in KpDyP Variants
3.4. The Distal Heme Side in KpDyP
4. Materials and Methods
4.1. Protein Preparation
4.2. Optical Absorption Spectroscopy
4.3. EPR Spectroscopy
- X-band continuous-wave (CW) EPR experiments were performed on a Bruker ESP300E spectrometer (Bruker Biospin, Rheinstetten, Germany) operating at a microwave frequency of ca. 9.44 GHz equipped with a liquid-helium cryostat (Oxford Inc., Oxford, UK) to enable temperatures from 2.5 K up to room temperature. Calibration of the magnetic field was done using a Bruker ER035M NMR Gaussmeter. The EPR tubes were vacuum-pumped to 1 mbar prior to and during the experiments to remove excess of paramagnetic dioxygen.
- X-band HYSCORE (hyperfine sublevel correlation spectroscopy) experiments [57] were carried out on a Bruker E580 Elexsys spectrometer (Bruker Biospin, Rheinstetten, Germany) (microwave frequency ≈ 9.74 GHz) equipped with an Oxford Instruments gas-flow cryogenic system to obtain an operating temperature of 4 K. The pulse sequence π/2-τ- π/2-t1-π-t2- π/2-τ-echo was performed using tπ/2 = 16 ns, tπ = 32 ns and was repeated for four different τ-values (96, 104, 114 and 128 ns) at a magnetic field corresponding to gzeff. Matched HYSCORE [58] was performed at a magnetic field corresponding to gyeff, using tπ/2 = 16 ns, tπ = 32 ns, tHTA = 24 ns and was repeated for two different τ-values (144 and 164 ns). t1 and t2 were varied in time steps of 16 ns, starting from 96 ns to 4896 ns. The HYSCORE spectra are baseline corrected using a third-order polynomial, apodized with a Hamming window and zero-filled. After Fourier transformation, the absolute value spectrum was calculated. HYSCORE measurements recorded with different τ-values were added together as indicated in the figure captions.
- W-band (93.98 GHz) electron spin echo (ESE)-detected EPR were performed on a Bruker Elexsys E680 spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a standard single-mode cylindrical resonator from Bruker and a continuous-flow cryostat and superconducting magnet from Oxford Instruments. Using a π/2-τ-π-τ-echo sequence, the low-field part (<1800 mT) was obtained using π/2 (π) pulse lengths of 60 (120) ns and τ = 200 ns. The high-field part, representative for the low-spin signals, required π/2 (π) pulse lengths of 80 (160) ns and τ = 400 ns.
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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gz | gy | gx | Ref. | |
---|---|---|---|---|
WT KpDyP | ||||
OH2 | 2.77 | 2.17 | 1.77 | this work |
D143A KpDyP | ||||
OH1′ | 2.89 | 2.12 | 1.78 | this work |
OH2′ | 2.728 | 2.15 | 1.772 | this work |
OH3′ | 2.67 | 2.214 | 1.82 | this work |
HRP | 2.94 | 2.08 | 1.63 | [30] |
CcP | 2.74 | 2.22 | 1.74 | [31] |
swMb | 2.55 | 2.17 | 1.85 | [29] |
CeGLB-33 | 2.62 2.845 | 2.20 2.12 | 1.815 1.69 | [33] |
TfHb | 2.73 2.66 2.82 | 2.19 2.19 2.32 | 1.76 1.81 1.60 | [32] |
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Nys, K.; Pfanzagl, V.; Roefs, J.; Obinger, C.; Van Doorslaer, S. In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol. Int. J. Mol. Sci. 2021, 22, 9849. https://doi.org/10.3390/ijms22189849
Nys K, Pfanzagl V, Roefs J, Obinger C, Van Doorslaer S. In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol. International Journal of Molecular Sciences. 2021; 22(18):9849. https://doi.org/10.3390/ijms22189849
Chicago/Turabian StyleNys, Kevin, Vera Pfanzagl, Jeroen Roefs, Christian Obinger, and Sabine Van Doorslaer. 2021. "In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol" International Journal of Molecular Sciences 22, no. 18: 9849. https://doi.org/10.3390/ijms22189849